Molecular signalling in plants In the last 20 years, nitric oxide (NO) has been identified as an important chemical messenger in plants. Using a combination of biochemical and bioinformatics approach EU-funded research has identified thousands of potential target proteins and specific binding sites, opening the door to tests of function. Health © Thinkstock Living organisms have a very complex network of biochemical signalling to carry out virtually all functions from the cellular level up to the level of the organism. NO plays a role in regulation of plant functions, including disease resistance, gas exchange, seed germination and root development. Many of the biological functions result from interactions of NO (or more generally, the nitric oxide family (NOx)) with proteins. EU-funded scientists launched the project 'NO-dependent protein translocation and S-nitrosylation of nuclear proteins in Arabidopsis thaliana' (PRONITROARAB) to determine targets that mediate effects with a focus on nuclear proteins. One of the most important ways NO regulates functions in plants is to attach itself covalently to cysteine (S) residues of other molecules (S-nitrosylation). First, the team employed computational methods (the GPS-SNO 1.0 software, a recently developed S-nitrosylation site prediction programme) to survey the entire proteome of A. thaliana (27 416 proteins). The results were impressive. Candidate S-nitrosylation proteins were found in all cellular compartments (e.g. membrane, chloroplast, etc.) in large quantities. When focusing on the most likely candidates (more stringent statistical probability), the team identified 3 190 total S-nitrosylation sites in a total of 3 005 target proteins, primarily in chloroplast, the intracellular compartment and plasmodesmata. They represented 5–17 % of total protein content per compartment. Then the team had a closer look on S-nitrosylation of nuclear proteins in-vivo. To find S-nitrosylated nuclear proteins that are related to plant defence response the team exposed A. thaliana to a pathogen. This small flowering plant is a model system in plant biology. Proteins were then extracted from the nuclei and subjected to the biotin switch assay to identified S-nitrosylated nuclear proteins. Nuclear extracts exposed to an NO donor were used as control. Of the 195 candidates identified, 57 % (111) were nuclear proteins. These proteins serve a myriad of functions demonstrating the extensive reach of the NO pathway in regulation. Understanding regulatory mechanisms in plants is important on a number of levels, from acquiring fundamental knowledge to applications related to crop growth or disease resistance, and in pointing to similar pathways in other systems. PRONITROARAB contributed new knowledge to the field of plant regulation by S-nitrosylation of proteins and opened the door to numerous experiments and a bright career investigating these phenomena. Keywords Plants, nitric oxide, S-nitrosylation, nuclear proteins, GSP-SNO 1.0 software
